High axial-resolution optical stimulation of neurons in vivo via two-photon optogenetics with speckle-free beaded-ring pattern

Two-photon optogenetics has become an indispensable technology in neuroscience, due to its capability in precise and specific manipulation of neural activities. Scanless holographic approach is generally adopted to meet the requirement of stimulating neural emsembles simultaneously. However, the commonly used disk patterns fail in achieving single-neuron resolution, especially in axial dimension, and its inherent speckles decrease the stimulation efficiency. Here, we propose a novel speckle-free, beaded-ring pattern for high axial-resolution optical stimulation of neurons in vivo. Using a dye pool and a fluorescent thin-film as samples, we verify that, compared to those with disk patterns, higher axial resolution and better localization ability can be achieved with beaded-ring patterns. Furthermore, we perform two-photon based all-optical physiology with neurons in mouse S1 cortex in vivo, and demonstrate that the axial resolution obtained by beaded-ring patterns can be improved by 24% when stimulating multiple neurons, compared with that of disk patterns.